PROJECT SUMMARY The goal of this R21 Exploratory/Developmental Research Grant) proposal is to determine the structural basis for ligand recognition and binding by the extraterminal (ET) domain of the bromodomain and extraterminal (BET) domain family of proteins (e.g., Brd2, 3, 4). This objective is significant because the ET domain is the center piece for protein-protein interactions (PPIs) that (i) recruit a variety of host factors to epigenetic markers on chromatin, and (ii) determine DNA integration sites of ?-retrovirus-based gene therapy vectors. We expect these developmental studies to lead to (i) discovery of new anti-cancer and anti-viral therapeutics that function by preventing PPIs that modulate epigenetic gene expression, and (ii) improved strategies for eliminating the oncogenic side effects of otherwise effective retrovirus-based gene therapy approaches. The proposal is inspired by the discovery that the BET proteins (Brd2, 3, 4) are the principal cellular binding partners of Moloney murine leukemia virus integrase (MLV IN), and play a central role in targeting MLV integration to transcription start sites. Moreover, we showed that a fragment derived from the extreme C-terminus of MLV IN is necessary and sufficient for specific interaction with the ET domain of Brd4. Thus, structural studies of the IN-ET complex will illuminate determinants for ligand recognition by the conserved ET domain, thereby revealing how this domain is able to recruit protein factors to chromatin. We hypothesize that targeting these interactions via site-directed mutagenesis and small molecule inhibitors will open new research directions in cancer treatment and gene therapy. The aims of the proposal are to (1) determine the three-dimensional solution structure of the complex between the Brd4 ET domain and the MLV IN ET-binding motif (EBM), (2) define the specificity determinants for Brd4 ET binding to viral and cellular factors via thermodynamic analysis and site-directed mutagenesis, and (3) assess the utility of EBM-inspired small molecules to inhibit BET-host factor interactions. Our structural studies in aim 1 will reveal how the ET domain recognizes its targets. In aim 2 we will complement the structural insights with thermodynamic data, while we test potential peptide ligands from host cofactors. Finally, we will use that knowledge to develop new model compounds to inhibit those protein-protein interactions and test their utility in vitro and in vivo The proposed studies will reveal the structural basis for ligand binding by the ET domain of BET proteins, and yield new reagents (PPI inhibitors, mutants) for probing transcriptional regulatory pathways. These will be useful for both identifying and validating native cellular cofactors, and for highlighting potential targets for transcription-targeted therapies. Moreover, knowledge of how ?-retroviruses recognize BET proteins will illuminate strategies for altering the chromosomal integration patterns of MLV-based vectors in order to avoid activation of proto-oncogenes.